Compelling evidence identifies a synergism between alcohol and HCV infection for the risk of developing hepatocellular carcinoma (HCC). This application seeks support for research directed to the role of Nanog-positive cancer stem cells in hepatocarcinogenesis induced by HCV and alcohol. A mechanistic link between alcohol and HCV has recently been unveiled by our discovery that HCV NS5A protein induces Toll-like receptor 4 (TLR4) in hepatocytes, which in turn mediates liver tumor development in mice fed alcohol. TLR4 signaling activated by alcohol-induced endotoxemia, induces the progenitor/stem marker Nanog and liver tumors in NS5A transgenic (Tg) mice but not in wild type (wt) or NS5A Tg mice deficient in TLR4. Nanog immunostaining is co-localized with the cancer stem cell marker CD133 and CD49f in liver tumors of these mice. In vitro promoter and mRNA analyses demonstrate TLR4-dependent transcriptional activation of Nanog in NS5A-transfected cells. Transplantation of p53-/- hepatoblasts transduced with TLR4, produces liver tumors in recipient mice after repetitive LPS injection, and this tumor formation is prevented with Nanog shRNA. Transplantation of Nanog+/CD133+/CD49f+ cells but not Nanog-/CD133-/CD49f+ cells isolated from liver tumors of our mouse model, causes tumor growth in nude mice given repetitive LPS injection. Lentiviral cDNA library established from the former cancer stem cells but not the latter cells, transforms the oval cell line in vitro. These findings support the hypothesis that synergistic liver oncogenesis by HCV/NS5A and alcohol is mediated by TLR4-induced oncogenic activity of Nanog+ cancer stem cells. To test this hypothesis and understand the mechanisms of oncogenesis in the model, we will pursue two major aims: 1) to identify and validate oncogenic genes in the Nanog+ cancer stem cells; and 2) to elucidate genetic and epigenetic mechanisms of TLR4-mediated Nanog induction in the cancer stem cells. For the Aim 1, a lentiviral cDNA library established from the cancer stem cells will be used to isolate candidate oncogenic genes; shRNA-based knockdown will be applied to validate these oncogenic genes in vitro and in vivo; and ChIP-seq analysis will be performed for genome-wide analysis for Nanog binding in the cancer stem cells. For the Aim 2, we will elucidate the transcriptional mechanisms by which TLR4 induces Nanog via promoter-reporter assay with deletion constructs and site-directed mutagenesis, and ChIP analysis; and determine epigenetic regulation for sustained Nanog induction via DNA methylation analysis and ChIP analysis for histone modifications. In summary, the proposed R01 application represents novel mechanistic research focused on Nanog+ liver cancer stem cells generated via activated TLR4 signaling by HCV NS5A and alcohol. An ultimate goal of this application is to eventually translate basic knowledge derived from the current study to design novel therapeutic modalities targeted to Nanog+ cancer stem cells for HCV/alcohol-related HCC. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is one of the most important causes of liver cancer, which is the third most deadly cancer in the world. The goal of this proposal is to understand how HCV and alcohol induces liver cancer so that better prevention and treatment can be found.